Pre-M/M epitopes of dengue virus, synthetic peptides, chimeric proteins and their use

ABSTRACT

The present invention relates to five synthetic peptides of pre-M/M protein of Dengue-2 virus, corresponding to amino acid sequences 3-31, 45-67, 57-92, 69-93 and 103-124. The anti-peptide immune response was evaluated in mice. Recombinant fusion proteins were also obtained, including regions of pre-M/M protein. The presence of B cell epitopes in both mice and humans was demonstrated in the pre-M/M protein peptides. Peptides 3-31 and 103-124 elicited neutralizing antibodies against the four serotypes of Dengue virus. Virus-specific proliferative responses were demonstrated in mice immunized with non-conjugated peptides 3-31 and 57-92. Mice immunized with conjugated peptides 3-31, 57-92, and 69-93 were protected when they were challenged with Dengue-2 virus. Thus, the presence of sequential epitopes in Pre-M/M protein of Dengue-2 virus was demonstrated, as well as their relevance in the immune response against this flavivirus.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a 371 national stage entry of PCT/CU98/00001 filedJan. 13, 1998.

FIELD OF THE INVENTION

The present invention is in the field of biotechnology and relates torecombinant DNA techniques, in particular to the production of syntheticpeptides coding for pre-M/M protein of Dengue virus serotype 2 andchimeric proteins which contain epitopes of pre-M/M protein of Denguevirus serotype 2 and 4.

The technical objective is to identify Pre-M/M neutralizing andprotective epitopes, cross reactive for all dengue virus serotypes toobtain an immunogen for human vaccination.

BACKGROUND

Dengue virus belongs to the Flavivirus genus, family Flaviviridae(Westaway, E. G. et al. 1985. Flaviviridae. Intervirol. 24 p.183). It isan enveloped virus with a single RNA chain of positive polarity asgenetic material, which codes for a polyprotein processed co- andpost-transductionally by cellular and viral proteases.

There are two structural proteins in the viral membrane: E (envelope)and M (membrane), while there are several copies of the other structuralprotein, C (capside) forming the isometric nucleocapside. Besides, atleast seven non-structural proteins have been identified (NS1, NS2a,NS2b, NS3, NS4a, NS4b, NS5).

Glycoproteins E and NS1 are individually able to offer active andpassive protection against the homologous serotype of Dengue virus,while the highly conformational complexity of the relevant epitopes ispreserved. For this reason, recombinant eukaryotic cellular systems havebeen mainly selected for the immunological evaluation of these proteins,for example vaccinia virus (Bray, M. et al. 1989. Mice immunized withrecombinant Vaccinia virus expressing dengue-4 structural proteins withor without nonstructural protein NS1 are protected against fatal denguevirus encephalitis. J. Virol. 63 p.2853) and baculovirus (Zhang, Y. M.et al. 1988. Immunization of mice with dengue structural proteins andnonstructural protein NS1 expressed by baculovirus recombinant inducesresistance to dengue virus encephalitis. J. Virol. 62 p.3027).

The small protein M (8 kDa) is synthesized like a glycosylated precursornamed pre-M (22 kDa approximately), which suffers a late endoproteoliticcleavage just before or after the liberation of the virus of theinfected cell (Murray, J. M. et al. 1993. Processing of the dengue virustype 2 proteins prM and C-prM. J. Gen. Virol. 74 p.175). The cleavage,which is probably done by a cellular protease, seems to happen in thepost-Golgi acidic vesicles, being inhibited by agents that destabilizedthe low pH of this vesicles (Randolph, V. B. et al. 1990. Adidotropicamines inhibit proteolytic processing of Flavivirus prM protein. Virol.174 p.450). The fragment pre- has been identified in vitro only in theextracellular medium, its destiny in vivo remains unknown (Murray, J. M.et al. 1993. Processing of the dengue virus type 2 proteins prM andC-prM. J. Gen. Virol. 74 p.175).

It is thought that the function of pre-M/M during the Flavivirusexocytic via is to avoid the activation of the fusogenic membrane domainof E with the acidic pH of the environment (Randolph, V. B. et al. 1990.Acidotropic amines inhibit proteolytic processing of Flavivirus prMprotein. Virol. 174 p.450); if this event happens, then the viralliberation will be prevented. In fact, it has been determined that pre-Mand E interact in the immature intracellular virions (Wengler, G. yWengler, G. 1989. Cell-associated West Nile flavivirus is covered withE+pre-M protein heterodimers which are destroyed and reorganized byproteolytic cleavage during virus release. J. Virol. 63 p.2521), andthat the native conformation of E it is only acquired in the presence ofpre-M (Konishi, E. y Mason, P. W. 1993. Proper maturation of theJapanese encephalitis virus envelope glycoprotein requires cosynthesiswith the premembrane protein. J. Virol. 67 p.1672). In addition, alreadyliberated virions that only have pre-M in their membranes show, ingeneral, a lower infectivity than the completely mature virion (Wengler,G. y Wengler, G. 1989. Cell-associated West Nile flavivirus is coveredwith E+pre-M protein heterodimers which are destroyed and reorganized byproteolytic cleavage during virus release. J. Virol. 63 p.2521), inwhich although M and pre-M are present, the former is predominant.

Pre-M and M offer an active protection when they have been expressed inrecombinant vaccinia virus, but this do not happen with the fragmentpre- (Bray, M. y Lai, C.-J. 1991. Dengue virus premembrane and membraneproteins elicit a protective immune response. Virol. 185 p.505), besidesthe combination pre-M or M with glycoprotein E in the same recombinantVaccinia virus gives in general levels of protection higher than thosereached by each protein individually. Similarly, certain antibodiesagainst pre-M/M are able to protect passively in mice (Kaufman, B. M. etal. 1989. Monoclonal antibodies for dengue virus prM glycoproteinprotect mice against lethal dengue infection. Am J. Trop. Med. & Hyg. 41p.576).

The use of synthetic peptides has allowed to establish the molecularbasis of antigenicity according to spacial conformation and theimmunological properties of the antigen involved [Arnon, R. y Sela, M.1985. Synthetic Vaccines: present and future. Ann. Inst. Pasteur/Immunol136 D, 271-282]. The synthetic peptides as anti-dengue vaccine subunitswill allow to include in the final formulation only the protectiveepitopes that do not cause immune-amplification (Halstead, S. B., yO'Ruourke, E. J. 1977. Dengue viruses and mononuclear phagocytes. I.Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146p.201; Halstead, S. B. 1979. In vivo enhancement of dengue virusinfection in rhesus monkeys by passively transferred antibody. J.Infect. Dis. 140 p.527), or altematively, to include protective peptidesof each of the four serotypes. The characterization of the antigenicdeterminants of E and NS1 has been carried out successfully. However,there are no similar studies on the also important protein pre-M/M, thatis why the results of this paper are a first step on that direction.

The efforts to express the flaviviral protein pre M, M and E in E. colihave not always been successful (Chambers, T. J. et al. 1990. Productionof yellow fever virus proteins in infected cells: identification ofdiscrete polyprotein species and analysis of cleavage kinetics usingregion-specific polyclonal antiserum. Virol. 177 p.159; Yan, B.-S. etal. 1994. Truncating the putative membrane association regioncircumvents the difficulty of expressing hepatitis C virus protein E1 inEscherichia coli. J. Virol. Meths. 49 p.343). Apparently, thehydrophobic regions these protein have in C-terminal are the cause ofthe low or undetectable heterologous expression levels (Yan, B.-S. etal. 1994. Truncating the putative membrane association regioncircumvents the difficulty of expressing hepatitis C virus protein E1 inEscherichia coli. J. Virol. Meths. 49 p.343).

The expression of those proteins (as well as NS1) in E.coli, in generalhave been obtained by fusion (fragmented or not) to other bacterialproteins (e.g. b -galactosidase (Cane, P. A. y Gould, E. A. 1988.Reduction of yellow fever mouse neurovirulence by immunization with abacterially synthesized non-structural protein (NS1) fragment. J. Gen.Virol. 69 p.1241), TRPE (Megret, F. et al. 1992. Use of recombinantfusion proteins and monoclonal antibodies to define linear anddiscontinuous antigenic sites on the Dengue envelope glycoprotein.Virol. 187 p.480) and the protein A of Staphylococcus aureus (Murray, J.M. et al. 1993. Processing of the dengue virus type 2 proteins prM andC-prM. J. Gen. Virol. 74 p.175). In these fusion proteins most of therelevant conformational epitopes are absent, because though the antiseragenerated against them can recognize the whole virus, they are not ableneither to neutralize it nor to inhibit their hemagglutinatingproperties (Megret, F. et al. 1992. Use of recombinant fusion proteinsand monoclonal antibodies to define linear and discontinuous antigenicsites on the Dengue envelope glycoprotein. Virol. 187 p.480). However,recent reports show that the solubility of the fusion proteins, and as aconsequence, the use of non-denaturalizing methods for its purification,may preserve most of the neutralizing (Seif, S. A. et al. 1995. Finermapping of neutralizing epitope(s) on the C-terminal of Japaneseencephalitis virus E-protein expressed in recombinant Escherichia colisystem. Vaccine 13 p.1515) and protective (Srivastava, A. K. et al.1995. Mice immunized with a dengue type 2 virus E and NS1 fusion proteinmade in Escherichia coli are protected against lethal dengue virusinfection. Vaccine 13 p.1251) epitopes they possess.

In the case of pre-M/M, its pre-domain has 6 cysteines involved in 3disulfide bridges, as well as an N-glycosylation site in the asparagine69. The structure of E and NS1 is even more complicated; it involves 6disulfide bridges and several N-glycosylation sites. However, the littleectodomain of M is apparently free of those conformational complexitiesbecause it does not have cysteines, and it is not glycosylated in itsnatural form.

The insertion of heterologous fragments in permissive areas ofimmunogenic proteins which topology is more or less known andimmunization of these fusions is a complementary alternative to the useof synthetic peptides. Both strategies allow defining the presence ofsequential B cell, as well as T cell epitopes. The biological importanceof these epitopes could be experimentally evaluated to decide where toinclude them or not in certain vaccine preparation.

SUMMARY OF THE INVENTION

The invention, provides synthetic peptides of Dengue virus preM/Mprotein comprising amino acids 3-11 (Sequence ID No.:1), 45-67 (SequenceID No.:2), 57-92 (Sequence ID No.:3) 69-93 (Sequence ID No.:4) and103-124 (Sequence ID No.:5) that include at least one opitope that iscross reactive with any Dengue virus serotype; and mimetic compoundsthereof.

Also provided are diagnostic tests and pharmaceutical formulations thatinclude the above peptides or mimetic compounds useful in the detectionand prophylaxis or treatment of flavivirus infections.

The invention further provides antibodies and antibody fragments thatspecifically bind the Dengue virus preM/M protein comprising amino acids3-31 (Sequence ID No.:1), 45-67 (Sequence ID No.:2), 57-92 (Sequence IDNo.:3), 69-93 (Sequence ID No.:4) and 103-124 (Sequence ID No.:5) thatinclude at least one epitope that is cross reactive with any Denguevirus scrotype. Vaccine and therapeutic preparations that include theseantibodies are also provided.

Further, the invention provides genetic constructs that include aminoacid sequences that include epitopes of pre-M/M from Dengue virusscrotypes 2 and 4, fused to a carrier protein.

DETAILED DESCRIPTION OF THE INVENTION

Five peptides from Pre-M/M protein of Dengue 2 virus which cover 58% ofthe aminoacid sequence (97/166 AA) were chemically synthesized. Theywere 3-31; 45-67; 57-92; 69-93; and 103-124, which were subsequentlynamed B 19-6; B 20-2; B 19-5; B 20-1; B 20-3 respectively.

Peptides were inoculated in Balb/c mice both conjugated or not to acarrier protein. The sera obtained after immunization with theconjugated peptides were tested by in vitro neutralization by reducingthe number of plaques and by ELISA. We also studied the activeprotection against a Dengue 2 viral challenge in the immunized mice.

In the case of mice immunized with the non-conjugated peptides, theantibody response was evaluated by ELISA and the proliferative responseof spleen T lymphocytes against Dengue 2 virus was evaluated too. Fusionproteins were also obtained, and two of the four regions covered bypeptides (1-42 and 92-133) were inserted to them and were expressed inE.coli bacteria. Immunization with these fusions will complement theresults obtained with the synthetic peptides.

The presence of B cell epitopes in both mice and humans was demonstratedas the peptides were recognized by antibodies from the immunized miceand by sera from patients who had the clinical and serological diagnosisof Dengue virus, using ELISA in both cases. Peptides 19-6 and 20-3 wereable to induce neutralizing antibody production against the four Denguevirus serotypes.

Virus-specific proliferative responses were demonstrated in miceimmunized with non-conjugated peptides 19-6 and 19-5. Mice immunizedwith conjugated peptides 19-6, 20-1, and 19-5 showed a statisticallysignificative level of protection when they were challenged with Dengue2 virus.

Thus, the presence of sequential epitopes in Pre-M/M protein of Denguevirus 2 was demonstrated, as well as their relevance in the immuneresponse against these flaviviruses.

EXAMPLES Example 1

Prediction of Antigenic Regions and of T-cells Epitopes of Pre-M/MProtein of Dengue Virus

Different theoretical methods were applied to predict the antigenicregions in the pre-M/M protein of D2 virus. These regions are those morelikely to be recognized by antibodies obtained against the viralproteins, as well as to generate antibodies that recognized the originalproteins. Some methods to predict T-cell epitopes were applied. Fiveinitial peptides that have possible B- and T-cell epitopes were found (4in pre- and 1 in M). The study of the antigenic structure of theseproteins and the experimental determination of possible immunologicallyimportant peptides was based on this finding.

1.1 Predictions of Humoral Amino Acid

Methods used to predict the antigenicity were based on the aminoacidicsequence, since neither the three dimensional structure of the pre-M/Mprotein of Dengue virus has been determined experimentally, nor is therea significant similarity at sequence level with any protein of knownthree dimensional structure.

The A 15 strain of Dengue 2 isolated in Cuba in 1981 (Kour, G. et al.1986. Hemorrhagic dengue in Cuba: history of an epidemic. Bull. P.A.H.O20 p.24) was used to accomplish this example. The potentially antigenicregions were selected according to the following criteria:

a) regions of high antigenic propension according to differentprediction methods based on hydrophilicity (Hoop, T. P. y Woods, K. R.1981. Prediction of protein antigenic determinants from amino acidsequences. Proc. Natl. Acad. Sci. USA 78 p.3824; Parker, J. M. R. et al.1986. New hydrophility sale derived from HPLC peptide retention data:correlation of predicted surface residues with antigenicity and X-rayderived accessible sites. Biochemistry 25 p.5425), flexibility (Karplus,P. A. y Schultz, G. E. 1985. Prediction of chain flexibility inproteins. A tool for the selection of peptide antigens.Naturwissenschaften 72 p.212) and accessibility (Emini, E. A. et al.1985. Induction of hepatitis A virus-neutralizing antibody by a virusspecific synthetic peptide. J. Virol. 55 p.836).

b) regions with high possibility of forming loops and turns according topredictions of secondary structure that use PHD (Rost, B. y Sander, C.1993. Prediction of protein secondary structure at better than 70%accuracy. J. Mol. Biol. 232 p.584; Rost, B. y Sander, C. 1994. Combiningevolutionary information and neural networks to predict proteinsecondary structure. Proteins 19 p.55; Rost, B. y Sander, C. 1994.Conservation and prediction of solvent accessibility in proteinfamilies. Proteins 20 p.216).

c) regions of high variability that include or notinsertion/suppressions in respect to other flaviviruses, as well aspotential regions of glycosylation in other flaviviruses that are usedor not in Dengue virus.

A. Antigenicity Profiles

FIG. 1 shows the profiles that are obtained when applying to the pre-and M segments 4 properties of the amino acids related to theantigenicity.

In the pre- region there are high hydrophilicity and accessibilityvalues in the regions that have the residues 6-9, 16-21, 28-31, 42-47,58-65 and 82-91. It is remarkable the existence of a vast hydrophobicregion between the residues 41-76, which corresponds to thetransmembrane helices that are thought not to be exposed to the immunesystem. In the little ectodomain of M (residues 1-40) the region ofmajor hydrophilicity/accessibility goes between 13-31 amino acids,especially at its beginning (AA 13-16).

B. Predictions of Secondary Structure

FIG. 2 shows the predictions of secondary structure and accessibility ofthe pre- and M segments according to PHD program. The results of thepredictions show that many potentially antigenic regions (according tothe profiles of FIG. 1) are predisposed to form loops/b-turns withexposed residues in the surface of the protein. It is predicted theformation of transmembrane helices for the region between aminoacids41-76 of protein M, and this matches with the hydrophobic character ofthis region and suggests that the antigenic peptides of M are mainly inthe ectodomain (1-40).

a. Alignments of sequences of protein pre- and M of Dengue and otherFlavivirus. Variability and glycosylation.

In general, regions that are not exposed to the solvent have a biggerconservation in the family of homologous proteins. Therefore, regions ofhigher variability have a higher probability to be exposed.

In the case of viruses, variability is also an escape mechanism for theimmunological pressure; of course this does not exclude that someconserved regions might be antigenic or that there could be conservedregions in the surface.

The analysis of sequences of regions pre- and M of 15 isolates of the 4serotypes of Dengue virus shows that at least the 69% of the residuesare strictly conserved. The more important variable residues are in thepositions 28-30, 55-59, 69-72 and 80-83 of pre-, as well as in 27-30 ofM. In general; these zones match the maximum of antigenic profiles ofFIG. 1.

The comparison of the sequences of these regions in more than 30flaviviral isolates shows that the region 1-33 of pre is highlyvariable, with possible loops predisposed to insertions/suppressions (inthe positions 8 and 30) and several potential sites of N-glycosylation.On the contrary, the variability is lower in the domain 33-91 of pre-;there are several positions strictly conserved in all flaviviruses, forexample: 6 cysteins forming of 3 disulfide bridges, at least 5 addresidues in the region 4045, as well as the basic sequence 87-91, afterwhich the endoproteolytic cleavage occurs just before or during theliberation of the mature virus. (FIG. 3)

Asn-69, conserved residue in the antigenic Dengue complex has the onlyN-glycosylation of pre-M/M protein of the complex. However, in theFlaviviridae family this region is in a possible exposed loop of highvariability. At the same time the pre-M/M residues of Dengue virus thatmatch the potential N-glycosylation sites in other Flavivirus (forexample, AA 14 in JE, SLE, MVE YF and AA 32 in LI, LAN, YF TBE) areb-turns close to zones considered as antigenic.

1.2 Prediction of T Cell Epitopes

The prediction was done by two independent methods: the Rothbard andTaylor pattern method (Rothbard, J. B. y Taylor, W. R. 1988. A sequencepattern common to T-cell epitopes. EMBO J. 7 p.93.) and thedetermination of fragments with propensity to form alpha-helixstructures (AMPHI 7 and 11) (Margalit, H. et al. 1987. Prediction ofimmunodominant helper T cell antigenic sites from the primary sequence.J. Immunol. 138 p.2213.). Results are shown in FIG. 4.

1.3 Peptides Proposed for the Identification of Relevant Epitopes

The determination of neutralizing and protective peptides in general isvery important for the development of more efficient vaccines, andpeptides from regions of high antigenic propension are very useful fortheir identification; especially of those of linear nature.

Table 1 shows a set of peptides that include regions predisposed to haveB and T cell epitopes (according to the several predictive methods usedin this example) of pre-M/M protein of D2 virus. If the validity of thatprediction is demonstrated experimentally, the immunological importantepitopes of each region will be placed accurately by the design of lowsize peptides in each of them.

TABLE 1 Antigenic peptides proposed in pre-M/M protein of Dengue virus.CODE SEQUENCE REGION B 19-6 (SEQ ID NO: 1) LTTRNGEPHMIVMRQEKGKSLLFKTGDGV3-31 B 20-2 (SEQ ID NO: 2) CEDTITYKCPLLRQNEPEDIDCW 45-67 B 19-5 (SEQ IDNO: 3) RQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRS 57-92 B 20-1 (SEQ ID NO: 4)NSTSTWVTYGTCTTTGEHRREKRSV 69-93 B 20-3 (SEQ ID NO:5)LETRTETWMSSEGAWKHAQRIE 103-124

Example 2

Chemical Synthesis of Oligopeptides and Oligonucleotides

1. Synthesis of Oligopeptides

All peptides were synthesized using a Boc-strategy in solid phase on thep-methyl-benzhydrilamine resin (resin MBHA, BACHEM, Switzerland).

The protected amino acids were offered by BACHEM. The protection ofreactive groups of the amino acid chain was: Arg (Tos), Asp (OBzl), Cys(4-Me-Bzl), Glu (OBzl), Lys (2-CI-Z), Trp (CHO), Tyr (Cl2-Bzl), Thr(Bzl). Asn, Gin and Pro were used without protection in the lateralchains.

Discarding of the Boc-amino protective group was carried out using 37.5%trifluoracetic acid in dichloromethane. Activation withdiisipropyl-carbodiimide (DIC) in situ was used for the couplingreaction of each residue, except for amino acids Asn and Gln, which wereactivated using DIC and 1-hydroxybenzotriazole in N,N-dimethylformide.

Final desprotection and peptide liberation of the resin was accomplishedin special equipment. The procedure used is known as Low-High HF.

During the first part of the procedure (Low HF), the protected-resinsystem was treated with HF (25%): DMS (65%): p-cresol (10%) during 120minutes at 0. The mixture was replaced by HF (25%): DMS (60%): EDT(10%): p-cresol (5%) in the case of Trp-containing peptides.Subsequently, the resin-peptide was washed several times with diethylether, dichloromethane and 2-propanol, and was vacuum-dried.

During the second part of the procedure (High HF), the resin-peptide wastreated with HF (90%): anizole (10%) during 60 minutes at 0.

The raw product was washed with ether, then was extracted with 30%acetic in water and finally was lyophilized.

Peptides were characterized by RP-HPLC in a BAKER C-18 (4.6×100 mm)column and by spectrometry of masses using FAB as ionization method in aJEOL HX-110 HF equipment.

The aminoacidic sequence as well as its location in the preM-M proteinof Dengue virus is shown in Table 1.

2.2. Synthesis of Oligonucleotides

Oligonucleotides were automatically synthesized in the equipment GeneAssembler Plus, according to the phosphoramidite method.

The sequence of the six oligonucleotides is shown in Table 2.

Table 2. Sites Xba I and EcoR I that are created in each end for itslater manipulation are underlined and double-underlined, respectively.The reading frame of the codified protein is defined by the triplets ofbases.

Oligonucleotide

position

Nucleotidic sequence 1) 5′ pre DEN-2 5′-TTT CTA GAT TTC CAT TTA ACC ACACGT T-3′ 2) 3′ pre DEN-2 5′-T TTC TAG ACC AAG GTC CAT GGC CAT GAG-3′ 3)5′ M DEN-2 5′-TTT CTA GAA TCA GTG GCA CTC GTT CCA CAT G-3′ 4) 3′ M DEN-25′-T TTC TAG AAA GCC TGG ATG TCT CAA GAT CCA-3′ 5) 5′ M DEN-4 5′-TTT CTAGAT TCA GTA GCT TTA ACA CCA C-3′ 6) 3′ M DEN-4 5′-T TGA ATT CGC GAA TCTTGG GTT TCT GAG-3′

Example 3

Coupling of Peptides to a Carrier Protein and Immunization Scheme

3.1 Coupling of Peptides to BSA

The coupling of peptides was performed as follows:

1. Activation of BSA: Drop by drop and with shaking, 80 ml of thebifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)5 m g/ml in dimethylformamide, were added to a dissolution of 2.8 mg ofbovine albumin fraction V (BSA) in 250 ml of PBS. Then, it was kept inagitation at room temperature during 30 minutes, and the mixture waspassed through a PD10 column.

2. Coupling of the peptide to the activated BSA: a dissolution of 1 mgof peptide dissolved in 300 ml of PBS was added to the activated BSAsolution drop by drop and with shaking. It was kept at room temperatureduring 3 hours, and concentration was determined by the Lowry method.

3.2 Immunization Scheme

The immunization scheme of peptides linked to BSA is the following:

FIG. 5 shows the proliferative response to Dengue 2 virus antigens ofspoeen T cell from peptide-immunized mice.

Male Balb/c mice of 4-6 weeks old were immunized with 50 mg of thepeptide-BSA conjugate intraperitoneally. In addition, two immunizationschemes were performed, one with BSA and the other with PBS. A total of4 inoculations were carried out, each of them 15 days apart. TheFreund's Complete Adjuvant was used in the first doses, and the Freund'sIncomplete Adjuvant in the others. A blood sample was extracted from theretro-orbital vein seven days after the last inoculation.

The obtained sera of each scheme were placed at −20 C. to be used later.

Example 4

In vitro Plaque-reduction Neutralization Test

Neutralization technique was carried out according to Morens (Morens, D.M. et al. 1985. Simplified plaque reduction neutralization assay fordengue viruses by semimicro methods in BHK-21 cells: Comparison of theBHK suspension test with standard plaque reduction neutralization. J.Clin. Microbiol. 22 p.250).

Dilutions of anti-peptide sera and of anti-BSA controls and negativesera from 1/10 to 1/640 were prepared. Each dilution of sera was putinto contact with a dilution of the virus (strain A 15 of Dengue 2)having 15-20 PFU/50 ml.

The mixture was incubated at 37 C. during 1 hour. A total of 50 ml ofeach mixture were added in triplicate to BHK-21 cells in plates of 24wells, and they were incubated in a CO2 incubator at 37 C. during 4hours. Then, 0.5 ml of carboxymethylcellulose-containing medium wereadded, and it was incubated again for several days considering the viralserotype used. After these days tinction and the count of lysis plaquesproduced by the virus were carried out.

Titer was expressed in each case as the dilution at which it wasobtained the 50% of plaque number reduction.

The results are shown in Table 3.

TABLE 3 PRNT of the anti-peptide sera against 19-6 and 20-3.Anti-peptide neutralization titer for each serotype D1 D2 D3 D4 B 19-61/100 1/180 1/60 1/160 B 20-3 1/110 1/80  1/80 1/80 

Example 5

Identification of T Cell Epitopes

The presence of T cell epitopes in the peptides of pre-M was evaluatedthroughout the study of anti-peptide antibody response elicited in freepeptides (non-conjugated) immunized mice. Primed animals demonstratedhigher serum antibody production in response to a booster dose ofantigen when compared to the response in naive animals. These resultsconfirm the existence of B cell epitopes in these peptides and show thatthese sequences contains T cell epitopes, which are able to stimulate Thactivity in vivo to improve the titers of antibody response.

Virus-specific proliferative responses of spleen T lymphocytes weredemonstrated in peptide immunized BALB/c mice. T cells from 19-6 and19-5 immunized mice proliferated in an in vitro blastogenesis assay whenthey were cultured with the dengue 2 virus. However, the 20-2 peptidedid not elicit a significant proliferative response against the virus.It could contain a T cell cryptic epitope, being recognized in the freeform of peptide but not like a result of the immunodominant epitopespresentation and processing of the virus in a natural infection.

Example 6

Protection Assay

Mice were challenged 7 days after the last immunization by intracranealinjection with an dilution of 1/2500 (corresponding to 100 LD50 lethaldoses) of live, mouse-adapted dengue-2 virus (strain A15). Mice wereobserved for up to 21 days for morbidity and mortality. Data were testedfor statistical significance using Fisher's test. The percent survivalin peptide immunized and control animals are shown in FIG. 6. The levelof protection induced for the peptides 19-5, 19-6 and 20-1 wasstatistically significant (p<0,05).

Example 7

Indirect ELISA to Detect Anti-peptide Antibodies

Human Sera

Peptides 19-6, 20-1, 20-2, 20-3 were fixed to the plates in a 10 m g/mlconcentration in coating buff they were incubated at 4 C. overnight Serawere added diluted 1/200 in PBS-Tween 20. Finally, total human/peroxidase anti-immunoglobuline conjugate was added, and subsequently,the substrate (orthophenylendiamine, H2O2, 0.05 M phosphate citratebuffer, pH 5) was added. The reading was carried out in an ELISA readerat 492 nm and the cut-off value for each peptide was determined.

The sera used were from subjects having viral clinical infection thatwas serologically diagnosed as Dengue by the techniques of Inhibition ofthe hemagglutination (Clarke, D. H. y Casals, J. 1958. Techniques forhemagglutination and hemagglutination—inhibition with Arthropod BorneVirus. Am. J. Trop. Med. Hyg. 7 p.561) and ELISA of inhibition (Vzquez,S., Fernndez, R. 1989. Utilizacin de un mtodo de ELISA de Inhibicin enel diagnstico serolgico de dengue. Rev. Cub. Med. Trop. 41(1) p18-26)for total anti-dengue antibodies.

The study induded 118 sera from patents of the epidemics occurred inCuba 1981, Panama 1994 and Costa Rica 1994. Dengue virus 2 was isolatedin these epidemics, besides serotype 1 and 4 in Costa Rica; they wereclassified according to the titers of inhibiting antibodies ofhemagglutination in cases of primary and secondary infections.

The 46.6% of the sera were positive to the 4 peptides used. Percentagesof positivity of 56.8%, 79.6%, 77.1% and 83.1% to peptides B 19-6, 20-1,20-2, and 20-3 were obtained respectively.

The average of the reactivity index, calculated by the optical densityquotient of the sample/cut-off value, for each peptide was 1.07, 1.52,1.57 and 1.49 for each peptide.

Mouse Sera

The indirect ELISA used was as described above but using an anti-mouseIg conjugated to peroxidase. Antibody titers obtained in theanti-peptides sera were generally above 1/10 000.

Example 8

Insertion of Pre-M/M Fragments in the P64k Protein of Neisseriameningitidis

In this example we expressed fragments of the pre-M/M protein of Dengue2 (A 15 strain) and of Dengue 4 (814669 strain) (Zhao, B. et al. 1986.Cloning full-length dengue type 4 viral DNA sequences: analysis of genescoding for structural proteins. Virol. 155 p.77) inserted in a N.meningitidis protein previously characterized in our group (Silva, R. etal. 1992. Nucleotide sequence coding for an outer membrane protein fromNeisseria meningitidis and use of said protein in vaccine preparations.European Patent 0 474 313, 1997): P64k, which have demonstrated to behighly immunogenic in several animal models. Besides, the level ofexpression of P64k in E. coli reaches more than 30% of the total proteinof the bacteria.

P64k protein (64 kDa) of dimeric nature, has two functional domains ineach subunit: one with lipoic acid-binding activity (1-100) and theother with lipoamide-dehydrogenase activity (117-594). Both have beenidentified as conformational domains relatively independent bycrystallography of X-rays (Li de la Sierra, l. et al. 1994.Crystallization and preliminary X-ray investigation of a recombinantouter membrane protein from Neisseria meningitidis. J. Mol. Biol. 235p.1154; Li de la Sierra, I. et al. 1997. Molecular structure of thelypoamide dehydrogenase domain of a surface antigen from Neisseriameningitidis. J. Mol. Biol. 269 p.129).

The former was selected (in the aminoacidic position 45) to perform theinsertions of fragments 1-42 and 92-133 of pre-M/M, because this littledomain is more exposed and does not seem to be involved indimer-formation. This suggested that the global structure of thechimeric protein with respect to the natural P64k would be less alteredthan if an insertion site were made in the domain 117-594, which, inaddition participates directly in the formation of the dimer.

The region coding for amino acids 44-53 (TLETDKATMD), which include theregion of lipoic acid binding of the P64k gene used in the production offusion proteins, was preliminary changed to TLDLEMD. This modificationwas carried out to avoid the recognition of P64k by the sera of patientshaving primary cirrhosis, who have self-antibodies against homologousepitopes present in the human dehydrolipoamide acetyltransferasemytochondrial (Tuaillon, N. et al. 1992. A lipoyl syntheticoctadecapeptide of dihydrolipoamide acetyl transferase specificallyrecognized by anti-M2 autoantibodies in primary biliary cirrhosis. J.Immunol. 148 p.445).

The strategy to produce the two clones is explained below:

Fragments Pre-2, M-2 and M-4 were amplified by Polymerase ChainReaction, using the combination of oligonucleotides 1 and 2, 3 and 4,and 5 and 6, respectively (see Table 2), and using pD-5 plasmid astemplate. This pD-5 plasmid includes a copy of preM/M gene from Dengue-2virus (strain A-15) cloned into pBluescript vector (stratagene). DNAbands obtained in each case (120 bp) were digested with Xba I (Pre-2 andM-2) or Xba I/EcoR I (M-4), and they were cloned into the correspondingsites that were artificially created in position 135-145 of P64k gene,cloned into vector pM-92. Besides, a chimeric clone that include M-2 andM-4 bands in the already mentioned sites Xba I and EcoR I was generatedby triple ligation. Recombinant clones bearing the inserts in the rightorientation were identified by restriction analysis and DNA-sequencing.

The fusion proteins generated by clones of Pre-2 (pD31) SEQ ID NO.:6,M-2 (pD30) SEQ ID NO.:7, M-2/M-4 (pD33) SEQ ID NO.:9, and M-4 (pD34) SEQID NO.:8 were expressed under the promoter of the tryptophan operon(ptrp) in the E. coli strain MM294 (F-end A1 hsdR17 (rk− mk+) sup E44thi-1 relA1? RfbD1? SpoT1?). All were obtained in the expected sizes andwith levels of expression up to 30% of the total proteins of thebacteria, though PD31 protein showed a high instability (FIG. 7). Allfusion proteins were recognized by some mouse monoclonal antibodiesanti-P64k in ELISA (data not shown) and Western blotting (FIG. 8), wherea remarkable degradation in the whole-cell extract was detected. Theamino acid sequence of these proteins is shown the Sequence List.

Mouse immunization with PD33 and PD34 fusion proteins semipurified by anon-denaturalizing protocol, has elicited high titers against them inELISA (up to 1/100 000), and at the same time antibodies with titers upto 1/4000 in ELISA against the synthetic peptides have been obtained.

BREIF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Hydrophylicity, accessibility and flexibility profiles ofpre-(A) and M (B) proteins of Dengue virus.

FIGS. 2A-2B. Prediction of the secondary structure and of accessibilityof pre (A) and M (protein). AA: amino acids. PHD sec: prediction ofsecondary structure (E=beta, H=helix, L=loop) P-3 acc. Prediction ofaccessibility (e=exposed, b=non-exposed). Sub sec (Sub acc) residues inwhich the prediction of secondary structure (accessibility) is 82.4%(70%) efficacious.

FIG. 3. Variability profiles of pre- and M proteins. Variability wascalculated considering 3 sets of Flavivirus sequences. Dengue: sequencesof 15 Dengue isolations. MBV: Flavivirus sequences transmitted bymosquitoes that include Dengue virus, Kunji, West Nile Virus, MurrayValley Encephalitis and Saint Louis Encephalitis. Flavivirus: sequencesof more than 30 different flaviviral isolations: (MBV+Yellow fever,Langat, Louping III and Tick-Borne Encephalitis).

FIGS. 4A-4B. Prediction of T cell epitopes of pre (A) and M (B)proteins: AMPHI 7 (11): prediction of amphipatic segments of 7 (11)residues, positive residues are the central amino acids of an amphipaticblock potentially antigenic. RT 4 (5): prediction of antigenic profilesof 4 (5) residues, positive residues are those who fulfill the profiles.

FIG. 5. Proliferative response to dengue 2 virus antigens (atconcentrations of 10, 20 and 40 m g/ml) of spleen T cells frompeptides-immunized mice 19-6, 19-5 and 20-2.

FIG. 6. Percent survival in peptide immunized and control animals. Thelevel of protection induced for the peptides 19-5, 19-6 and 20-1 wasstatistically significant.

FIG. 7. 10% SDS-PAGE of MM294 E. coli strain transformed with fusionproteins and P64k protein (pM-92 plasmid). Lanes: 1-Untransformed MM294strain, 2-pM-92/MM294, 3-pD-30/MM294, 4-pD-31/MM294, 5-pD-33/MM294,6-pD-34/MM294.

FIG. 8. Western blot using AcM 114 of MM294 E. coli strain transformedwith fusion proteins and P64k protein (pM-92 plasmid). Lanes:1-Untransformed MM294 strain, 2-pM-92/MM294, 3-pD-30/MM294,4-pD-31/MM294, 5-pD-33/MM294, 6-pD-34/MM294.

9 1 29 PRT Dengue virus 1 Leu Thr Thr Arg Asn Gly Glu Pro His Met IleVal Met Arg Gln Glu 1 5 10 15 Lys Gly Lys Ser Leu Leu Phe Lys Thr GlyAsp Gly Val 20 25 2 23 PRT Dengue virus 2 Cys Glu Asp Thr Ile Thr TyrLys Cys Pro Leu Leu Arg Gln Asn Glu 1 5 10 15 Pro Glu Asp Ile Asp CysTrp 20 3 36 PRT Dengue virus 3 Arg Gln Asn Glu Pro Glu Asp Ile Asp CysTrp Cys Asn Ser Thr Ser 1 5 10 15 Thr Trp Val Thr Tyr Gly Thr Cys ThrThr Thr Gly Glu His Arg Arg 20 25 30 Glu Lys Arg Ser 35 4 25 PRT Denguevirus 4 Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Thr Gly1 5 10 15 Glu His Arg Arg Glu Lys Arg Ser Val 20 25 5 22 PRT Denguevirus 5 Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys1 5 10 15 His Ala Gln Arg Ile Glu 20 6 635 PRT Artificial SequenceDescription of Artificial Sequence Fusion Protein of Dengue virus preM/Mand Neisseria meningitidis P64k protein. 6 Met Ala Leu Val Glu Leu LysVal Pro Asp Ile Gly Gly His Glu Asn 1 5 10 15 Val Asp Ile Ile Ala ValGlu Val Asn Val Gly Asp Thr Ile Ala Val 20 25 30 Asp Asp Thr Leu Ile ThrLeu Asp Leu Asp Phe His Leu Thr Thr Arg 35 40 45 Asn Gly Glu Pro His MetIle Val Ser Arg Gln Glu Lys Gly Lys Ser 50 55 60 Leu Leu Phe Lys Thr GlyAsp Gly Val Asn Met Cys Thr Leu Met Ala 65 70 75 80 Met Asp Leu Gly LeuGlu Met Asp Val Pro Ala Glu Val Ala Gly Val 85 90 95 Val Lys Glu Val LysVal Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 100 105 110 Leu Ile Val ValVal Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 115 120 125 Glu Ala AlaAla Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 130 135 140 Ala ProGln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 145 150 155 160Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 165 170175 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 180185 190 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu195 200 205 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala AlaAsn 210 215 220 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met LeuArg Ala 225 230 235 240 Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly GlyLeu Ala Gly Met 245 250 255 Ala Lys Ser Arg Lys Val Asp Val Ile Gln GlyAsp Gly Gln Phe Leu 260 265 270 Asp Pro His His Leu Glu Val Ser Leu ThrAla Gly Asp Ala Tyr Glu 275 280 285 Gln Ala Ala Pro Thr Gly Glu Lys LysIle Val Ala Phe Lys Asn Cys 290 295 300 Ile Ile Ala Ala Gly Ser Arg ValThr Lys Leu Pro Phe Ile Pro Glu 305 310 315 320 Asp Pro Arg Ile Ile AspSer Ser Gly Ala Leu Ala Leu Lys Glu Val 325 330 335 Pro Gly Lys Leu LeuIle Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 340 345 350 Gly Thr Val TyrSer Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 355 360 365 Met Asp GlyLeu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 370 375 380 Gln LysGln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 385 390 395 400Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 405 410415 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 420425 430 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala435 440 445 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys GlnMet 450 455 460 Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile ValGly Gln 465 470 475 480 Pro Met Leu Ala His Lys Ala Val His Glu Gly HisVal Ala Ala Glu 485 490 495 Asn Cys Ala Gly His Lys Ala Tyr Phe Asp AlaArg Val Ile Pro Gly 500 505 510 Val Ala Tyr Thr Ser Pro Glu Val Ala TrpVal Gly Glu Thr Glu Leu 515 520 525 Ser Ala Lys Ala Ser Gly Arg Lys IleThr Lys Ala Asn Phe Pro Trp 530 535 540 Ala Ala Ser Gly Arg Ala Ile AlaAsn Gly Cys Asp Lys Pro Phe Thr 545 550 555 560 Lys Leu Ile Phe Asp AlaGlu Thr Gly Arg Ile Ile Gly Gly Gly Ile 565 570 575 Val Gly Pro Asn GlyGly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 580 585 590 Glu Met Gly CysAsp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 595 600 605 Pro Thr LeuGly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 610 615 620 Thr CysThr Asp Leu Pro Pro Gln Lys Lys Lys 625 630 635 7 635 PRT ArtificialSequence Description of Artificial Sequence Fusion Protein of Denguevirus preM/M and Neisseria meningitidis P64k protein. 7 Met Ala Leu ValGlu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn 1 5 10 15 Val Asp IleIle Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val 20 25 30 Asp Asp ThrLeu Ile Thr Leu Asp Leu Glu Ser Val Ala Leu Val Pro 35 40 45 His Val GlyMet Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser 50 55 60 Glu Gly AlaTrp Lys His Ala Gln Arg Ile Glu Thr Trp Ile Leu Arg 65 70 75 80 His ProGly Phe Leu Glu Met Asp Val Pro Ala Glu Val Ala Gly Val 85 90 95 Val LysGlu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 100 105 110 LeuIle Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 115 120 125Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 130 135140 Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 145150 155 160 Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala PheAla 165 170 175 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg TyrLys Thr 180 185 190 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro SerLys Ala Leu 195 200 205 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg HisLeu Ala Ala Asn 210 215 220 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp IleAsp Met Leu Arg Ala 225 230 235 240 Tyr Lys Asp Gly Val Val Ser Arg LeuThr Gly Gly Leu Ala Gly Met 245 250 255 Ala Lys Ser Arg Lys Val Asp ValIle Gln Gly Asp Gly Gln Phe Leu 260 265 270 Asp Pro His His Leu Glu ValSer Leu Thr Ala Gly Asp Ala Tyr Glu 275 280 285 Gln Ala Ala Pro Thr GlyGlu Lys Lys Ile Val Ala Phe Lys Asn Cys 290 295 300 Ile Ile Ala Ala GlySer Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 305 310 315 320 Asp Pro ArgIle Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 325 330 335 Pro GlyLys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 340 345 350 GlyThr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 355 360 365Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 370 375380 Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 385390 395 400 Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr PheGlu 405 410 415 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala ValLeu Val 420 425 430 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser AlaGlu Lys Ala 435 440 445 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu ValAsp Lys Gln Met 450 455 460 Arg Thr Asn Val Pro His Ile Tyr Ala Ile GlyAsp Ile Val Gly Gln 465 470 475 480 Pro Met Leu Ala His Lys Ala Val HisGlu Gly His Val Ala Ala Glu 485 490 495 Asn Cys Ala Gly His Lys Ala TyrPhe Asp Ala Arg Val Ile Pro Gly 500 505 510 Val Ala Tyr Thr Ser Pro GluVal Ala Trp Val Gly Glu Thr Glu Leu 515 520 525 Ser Ala Lys Ala Ser GlyArg Lys Ile Thr Lys Ala Asn Phe Pro Trp 530 535 540 Ala Ala Ser Gly ArgAla Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 545 550 555 560 Lys Leu IlePhe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 565 570 575 Val GlyPro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 580 585 590 GluMet Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 595 600 605Pro Thr Leu Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 610 615620 Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 625 630 635 8 677 PRTArtificial Sequence Description of Artificial Sequence Fusion Protein ofDengue virus preM/M and Neisseria meningitidis P64k protein. 8 Met AlaLeu Val Glu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn 1 5 10 15 ValAsp Ile Ile Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val 20 25 30 AspAsp Thr Leu Ile Thr Leu Asp Leu Asp Phe His Leu Thr Thr Arg 35 40 45 AsnGly Glu Pro His Met Ile Val Ser Arg Gln Glu Lys Gly Lys Ser 50 55 60 LeuLeu Phe Lys Thr Gly Asp Gly Val Asn Met Cys Thr Leu Met Ala 65 70 75 80Met Asp Leu Gly Ser Val Ala Leu Val Pro His Val Gly Met Gly Leu 85 90 95Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys His 100 105110 Ala Gln Arg Ile Glu Thr Trp Ile Leu Arg His Pro Gly Phe Leu Glu 115120 125 Met Asp Val Pro Ala Glu Val Ala Gly Val Val Lys Glu Val Lys Val130 135 140 Lys Val Gly Asp Lys Ile Ser Glu Gly Gly Leu Ile Val Val ValGlu 145 150 155 160 Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala Glu Ala AlaAla Ala Pro 165 170 175 Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro Ala ProGln Ala Ala Gln 180 185 190 Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp ValVal Val Leu Gly Gly 195 200 205 Gly Pro Gly Gly Tyr Ser Ala Ala Phe AlaAla Ala Asp Glu Gly Leu 210 215 220 Lys Val Ala Ile Val Glu Arg Tyr LysThr Leu Gly Gly Val Cys Leu 225 230 235 240 Asn Val Gly Cys Ile Pro SerLys Ala Leu Leu His Asn Ala Ala Val 245 250 255 Ile Asp Glu Val Arg HisLeu Ala Ala Asn Gly Ile Lys Tyr Pro Glu 260 265 270 Pro Glu Leu Asp IleAsp Met Leu Arg Ala Tyr Lys Asp Gly Val Val 275 280 285 Ser Arg Leu ThrGly Gly Leu Ala Gly Met Ala Lys Ser Arg Lys Val 290 295 300 Asp Val IleGln Gly Asp Gly Gln Phe Leu Asp Pro His His Leu Glu 305 310 315 320 ValSer Leu Thr Ala Gly Asp Ala Tyr Glu Gln Ala Ala Pro Thr Gly 325 330 335Glu Lys Lys Ile Val Ala Phe Lys Asn Cys Ile Ile Ala Ala Gly Ser 340 345350 Arg Val Thr Lys Leu Pro Phe Ile Pro Glu Asp Pro Arg Ile Ile Asp 355360 365 Ser Ser Gly Ala Leu Ala Leu Lys Glu Val Pro Gly Lys Leu Leu Ile370 375 380 Ile Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Thr Val Tyr SerThr 385 390 395 400 Leu Gly Ser Arg Leu Asp Val Val Glu Met Met Asp GlyLeu Met Gln 405 410 415 Gly Ala Asp Arg Asp Leu Val Lys Val Trp Gln LysGln Asn Glu Tyr 420 425 430 Arg Phe Asp Asn Ile Met Val Asn Thr Lys ThrVal Ala Val Glu Pro 435 440 445 Lys Glu Asp Gly Val Tyr Val Thr Phe GluGly Ala Asn Ala Pro Lys 450 455 460 Glu Pro Gln Arg Tyr Asp Ala Val LeuVal Ala Ala Gly Arg Ala Pro 465 470 475 480 Asn Gly Lys Leu Ile Ser AlaGlu Lys Ala Gly Val Ala Val Thr Asp 485 490 495 Arg Gly Phe Ile Glu ValAsp Lys Gln Met Arg Thr Asn Val Pro His 500 505 510 Ile Tyr Ala Ile GlyAsp Ile Val Gly Gln Pro Met Leu Ala His Lys 515 520 525 Ala Val His GluGly His Val Ala Ala Glu Asn Cys Ala Gly His Lys 530 535 540 Ala Tyr PheAsp Ala Arg Val Ile Pro Gly Val Ala Tyr Thr Ser Pro 545 550 555 560 GluVal Ala Trp Val Gly Glu Thr Glu Leu Ser Ala Lys Ala Ser Gly 565 570 575Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp Ala Ala Ser Gly Arg Ala 580 585590 Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr Lys Leu Ile Phe Asp Ala 595600 605 Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile Val Gly Pro Asn Gly Gly610 615 620 Asp Met Ile Gly Glu Val Cys Leu Ala Ile Glu Met Gly Cys AspAla 625 630 635 640 Ala Asp Ile Gly Lys Thr Ile His Pro His Pro Thr LeuGly Glu Ser 645 650 655 Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr CysThr Asp Leu Pro 660 665 670 Pro Gln Lys Lys Lys 675 9 635 PRT ArtificialSequence Description of Artificial Sequence Fusion Protein of Denguevirus preM/M and Neisseria meningitidis P64k protein. 9 Met Ala Leu ValGlu Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn 1 5 10 15 Val Asp IleIle Ala Val Glu Val Asn Val Gly Asp Thr Ile Ala Val 20 25 30 Asp Asp ThrLeu Ile Thr Leu Asp Leu Glu Ser Val Ala Leu Thr Pro 35 40 45 His Ser GlyMet Gly Leu Glu Thr Arg Ala Glu Thr Trp Met Ser Ser 50 55 60 Glu Gly AlaTrp Lys His Ala Gln Arg Val Glu Ser Trp Ile Leu Arg 65 70 75 80 Asn ProArg Phe Leu Glu Met Asp Val Pro Ala Glu Val Ala Gly Val 85 90 95 Val LysGlu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 100 105 110 LeuIle Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 115 120 125Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 130 135140 Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 145150 155 160 Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala PheAla 165 170 175 Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg TyrLys Thr 180 185 190 Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro SerLys Ala Leu 195 200 205 Leu His Asn Ala Ala Val Ile Asp Glu Val Arg HisLeu Ala Ala Asn 210 215 220 Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp IleAsp Met Leu Arg Ala 225 230 235 240 Tyr Lys Asp Gly Val Val Ser Arg LeuThr Gly Gly Leu Ala Gly Met 245 250 255 Ala Lys Ser Arg Lys Val Asp ValIle Gln Gly Asp Gly Gln Phe Leu 260 265 270 Asp Pro His His Leu Glu ValSer Leu Thr Ala Gly Asp Ala Tyr Glu 275 280 285 Gln Ala Ala Pro Thr GlyGlu Lys Lys Ile Val Ala Phe Lys Asn Cys 290 295 300 Ile Ile Ala Ala GlySer Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 305 310 315 320 Asp Pro ArgIle Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 325 330 335 Pro GlyLys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 340 345 350 GlyThr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 355 360 365Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 370 375380 Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 385390 395 400 Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr PheGlu 405 410 415 Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala ValLeu Val 420 425 430 Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser AlaGlu Lys Ala 435 440 445 Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu ValAsp Lys Gln Met 450 455 460 Arg Thr Asn Val Pro His Ile Tyr Ala Ile GlyAsp Ile Val Gly Gln 465 470 475 480 Pro Met Leu Ala His Lys Ala Val HisGlu Gly His Val Ala Ala Glu 485 490 495 Asn Cys Ala Gly His Lys Ala TyrPhe Asp Ala Arg Val Ile Pro Gly 500 505 510 Val Ala Tyr Thr Ser Pro GluVal Ala Trp Val Gly Glu Thr Glu Leu 515 520 525 Ser Ala Lys Ala Ser GlyArg Lys Ile Thr Lys Ala Asn Phe Pro Trp 530 535 540 Ala Ala Ser Gly ArgAla Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 545 550 555 560 Lys Leu IlePhe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 565 570 575 Val GlyPro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 580 585 590 GluMet Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 595 600 605Pro Thr Leu Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 610 615620 Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 625 630 635

What is claimed is:
 1. An isolated peptide or mimetic compoundcomprising a sequence of no more than about 40 amino acids from Denguevirus preM/M, wherein the sequence of amino acids from Dengue viruspreM/M comprises an epitope found within a sequence selected from thegroup consisting of SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, SEQ IDNo.: 4 and SEQ ID No.:
 5. 2. The isolated peptide according to claim 1,wherein the amino acid sequence of Dengue virus pre-M/M is of a firstserotype; and wherein the amino acid sequence of Dengue virus pre-M/M ofthe first serotype includes an epitope that is cross-reactive withDengue virus pre-M/M of a second serotype.
 3. The isolated peptide ormimetic compound according to claim 2, wherein the amino acid sequenceof Dengue virus pre-M/M is cross-reactive with Dengue virus pre-M/M ofmore than one serotype.
 4. The isolated peptide or mimetic compoundaccording to claim 1, wherein the peptide or mimetic compound elicits aproliferative response of spleen T lymphocytes against Dengue 2 virus.5. The isolated peptide or mimetic compound according to claim 1,wherein the peptide or mimetic compound elicits an antibody in a mammal.6. The isolated peptide or mimetic compound according to claim 5,wherein the antibody elicited is protective against Dengue virusinfection.